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Protein dynamics by NMR has been reviewed extensively in recent years. These surveys show decisively that information on structure should be complemented by information on motion both to properly characterize the protein, and to understand its function. The time scale accessible by NMR extends from picoseconds to days, with different methods accessing different parts of this time axis. Here we focus on heteronuclear NMR spin relaxation used to study ps to ns protein dynamics. The slow limit of this time regime is determined by the global tumbling of the protein, with the rates for internal motion of the probe being typically faster. Based on experience gained over nearly a decade we came to the conclusion that the traditional method of NMR spin relaxation analysis in proteins and nucleic acids, called “model-free” (MF), does not extract adequately and fully the information inherent in the experimental data largely because it is oversimplified. We have developed an approach that overcomes many of the MF deficiencies. This method, called the slowly relaxing local structure (SRLS) may be regarded as a generalization of MF. SRLS predates the MF approach, and even provided derivations of the exact equivalents of the MF equations . The issues brought up above will be addressed in detail in this review. It will be shown that analogous, but physically distinct, SRLS and MF analyses often yield substantially different results, indicating that the oversimplifications inherent in MF have unfavorable practical implications. Within a broader perspective, we illustrate the disadvantages of applying parameterization instead of setting forth models, using mathematical instead of physical parameter definitions, and not abiding by the assumptions underlying the various equations used. We offer the concepts that underlie SRLS as an alternative to the model-free point-of-view, and we describe and illustrate how SRLS can be implemented in a practical fashion. We also indicate how improvements to the current SRLS approach can be introduced

We developed in recent years the slowly relaxing local structure (SRLS) approach for analyzing NMR spin relaxation in proteins. SRLS is a two-body coupled rotator model which accounts rigorously for mode-coupling between the global motion of the protein and the local motion of the spin-bearing probe and allows for general properties of the second rank tensors involved. We showed that a general tool of data analysis requires both capabilities. Several important functionalities were missing in our previous implementations of SRLS in data fitting schemes, and in some important cases, the calculations were tedious. Here we present a general implementation which allows for asymmetric local and global diffusion tensors, distinct local ordering and local diffusion frames, and features a rhombic local potential which includes Wigner matrix element terms of ranks 2 and 4. A recently developed hydrodynamics-based approach for calculating global diffusion tensors has been incorporated into the data-fitting scheme. The computational efficiency of the latter has been increased significantly through object-oriented programming within the scope of the C++ programming language, and code parallelization. A convenient graphical user interface is provided. Currently autocorrelated (15)N spin relaxation data can be analyzed effectively. Adaptation to any autocorrelated and cross-correlated relaxation analysis is straightforward. New physical insight is gleaned on largely preserved local structure in solution, even in chain segments which experience slow local motion. Prospects associated with improved dynamic models, and new applications made possible by the current implementation of SRLS, are delineated.

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An integrated computational methodology for interpreting NMR spin relaxation in proteins has been developed. It combines a two-body coupled-rotator stochastic model with a hydrodynamicsbased approach for protein diffusion, together with molecular dynamics based calculations for the evaluation of the coupling potential of mean force. The method is applied to 15 This study sets forth an integrated computational approach for treating NMR relaxation in proteins by combining stochastic modeling and molecular dynamics. The approach developed provides new insights by its application to a protein that experiences complex dynamics.

We investigate ps-ns dynamics of the Rho-GTPase Binding Domain (RBD) of plexin-B1, which plays a key role in plexin-mediated cell signaling. Backbone 15N relaxation data of the dimeric RBD are analyzed with the model-free (MF) method, and with the slowly relaxing local structure/molecular dynamics (SRLS-MD) approach. Independent analysis of the MD trajectories, based on the MF paradigm, is also carried out. MF is a widely popular and simple method, SRLS is a general approach, and SRLS-MD is an integrated approach we developed recently. Corresponding parameters from the RBD dimer, a previously studied RBD monomer mutant, and the previously studied complex of the latter with the GTPase Rac1, are compared. The L2, L3 and L4 loops of the plexin-B1 RBD are involved in interactions with other plexin domains, GTPase binding, and RBD dimerization, respectively. Peptide groups in the loops of both the monomeric and dimeric RBD are found to experience weak and moderately asymmetric local ordering centered approximately at the Ci-1α-Ciα axes, and ns backbone motion. Peptide groups in the α-helices and the β-strands of the dimer (the β-strands of the monomer) experience strong and highly asymmetric local ordering centered approximately at the Ci-1α-Ciα axes (N–H bonds). N–H fluctuations occur on the ps time-scale. An allosteric pathway for GTPase binding, providing new insights into plexin function, is delineated.

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